Untersuchungen zur Pea-CAH-I-like Immunreaktivität im Nervensystem von Schaben

Ein polyklonales Antiserum (AS) gegen das Peptid Pea-CAH-I aus den glandulären Zellen der Corpora cardiaca der Schabe Periplaneta americana markierte auch Antigene im Nervensystem (NS) von P.a. und anderen Insekten. Dieses AS wurde zur Differenzierung der Pea-CAH-I-like Immunreaktivität im NS und zur Isolierung kreuzreagierender Pea-CAH-I-ähnlicher Peptide eingesetzt. Dazu wurden verschiedene competitive und nichtcompetitive Enzymimmunoassays mit diesem AS entwickelt. Es konnte gezeigt werden, daß im NS offensichtlich kein Pea-CAH-I vorkommt. Die Immunreaktivität in fraktionierten Gewebeextrakten wurde mit dem competitiven Anti-Pea-CAH-I-Enzymimmunoassay, mit HPLC und MALDI-TOF-Massenspektrometrie untersucht. Im Retrocerebralkomplex (RCK) von P.a. wurden drei unbekannte Pea-CAH-I-ähnliche Peptide gefunden und im 5. Abdominalganglion von P.a. sowie im Gehirn und im RCK von Nauphoeta cinerea wurde das Peptid Pea-MIP identifiziert

In situ Formation of Polymer Microparticles in Bacterial Nanocellulose Using Alternative and Sustainable Solvents to Incorporate Lipophilic Drugs

Bacterial nanocellulose has been widely investigated in drug delivery, but the incorporation of lipophilic drugs and controlling release kinetics still remain a challenge. The inclusion of polymer particles to encapsulate drugs could address both problems but is reported sparely. In the present study, a formulation approach based on in situ precipitation of poly(lactic-co-glycolic acid) within bacterial nanocellulose was developed using and comparing the conventional solvent N -methyl-2-pyrrolidone and the alternative solvents poly(ethylene glycol), Cyrene TM and ethyl lactate. Using the best-performing solvents N -methyl-2-pyrrolidone and ethyl lactate, their fast diffusion during phase inversion led to the formation of homogenously distributed polymer microparticles with average diameters between 2.0 and 6.6 µm within the cellulose matrix. Despite polymer inclusion, the water absorption value of the material still remained at ~50% of the original value and the material was able to release 32 g/100 cm 2 of the bound water. Mechanical characteristics were not impaired compared to the native material. The process was suitable for encapsulating the highly lipophilic drugs cannabidiol and 3-O-acetyl-11-keto-β-boswellic acid and enabled their sustained release with zero order kinetics over up to 10 days. Conclusively, controlled drug release for highly lipophilic compounds within bacterial nanocellulose could be achieved using sustainable solvents for preparation

In Situ Formation of Polymer Microparticles in Bacterial Nanocellulose Using Alternative and Sustainable Solvents to Incorporate Lipophilic Drugs

Bacterial nanocellulose has been widely investigated in drug delivery, but the incorporation of lipophilic drugs and controlling release kinetics still remain a challenge. The inclusion of polymer particles to encapsulate drugs could address both problems but is reported sparely. In the present study, a formulation approach based on in situ precipitation of poly(lactic-co-glycolic acid) within bacterial nanocellulose was developed using and comparing the conventional solvent N-methyl-2-pyrrolidone and the alternative solvents poly(ethylene glycol), CyreneTM and ethyl lactate. Using the best-performing solvents N-methyl-2-pyrrolidone and ethyl lactate, their fast diffusion during phase inversion led to the formation of homogenously distributed polymer microparticles with average diameters between 2.0 and 6.6 µm within the cellulose matrix. Despite polymer inclusion, the water absorption value of the material still remained at ~50% of the original value and the material was able to release 32 g/100 cm2 of the bound water. Mechanical characteristics were not impaired compared to the native material. The process was suitable for encapsulating the highly lipophilic drugs cannabidiol and 3-O-acetyl-11-keto-β-boswellic acid and enabled their sustained release with zero order kinetics over up to 10 days. Conclusively, controlled drug release for highly lipophilic compounds within bacterial nanocellulose could be achieved using sustainable solvents for preparation

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stemcells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membraneprotrusions. Here, we investigate its impact on the architecture of membrane protrusions usingmicrovilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysisrevealed that upon the overexpression of prominin-1 the number of microvilli and clusters ofthem increased. Microvilli with branched and/or knob-like morphologies were observed andstimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypeswere caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 com-plex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibitedthe aforementioned interactions abolishing altered microvilli. This suggests that the interplay ofprominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton com-ponents regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutantsmodified the structure of filopodia emerging from fibroblast-like cells and silencing humanprominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated micro-villi. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellularprotrusions.Fil: Thamm, Kristina. Technische Universität Dresden; AlemaniaFil: Šimaitė, Deimantė. Technische Universität Dresden; AlemaniaFil: Karbanová, Jana. Technische Universität Dresden; AlemaniaFil: Bermúdez, Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Technische Universität Dresden; AlemaniaFil: Reichert, Doreen. Technische Universität Dresden; AlemaniaFil: Morgenstern, Anne. Technische Universität Dresden; AlemaniaFil: Bornhäuser, Martin. University Hospital Carl Gustav Carus; AlemaniaFil: Huttner, Wieland B.. Max Planck Institute of Molecular Cell Biology and Genetics; AlemaniaFil: Wilsch Bräuninger, Michaela. Max Planck Institute of Molecular Cell Biology and Genetics; AlemaniaFil: Corbeil, Denis. Technische Universität Dresden; Alemani

Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors

Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, i.e. Small Angle Neutron Scattering (SANS) and X-ray Scattering (SAXS), with cryo-Electron Transmission Microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments, suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structure consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insights, also combined with size distribution and zeta potential of the NPs, provides exaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development

Molecular Regulation of Acute Tie2 Suppression in Sepsis

Objectives: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness. Design: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients. Setting: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen. Patients: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22). Interventions: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration. Measurements and Main Results: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor--treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p <0.005). Conclusions: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript

Preparation of PEGylated liposomes incorporating lipophilic lomeguatrib derivatives for the sensitization of chemo-resistant gliomas

Liposomal delivery is a well-established approach to increase the therapeutic index of drugs, mainly in the field of cancer chemotherapy. Here, we report the preparation and characterization of a new liposomal formulation of a derivative of lomeguatrib, a potent O6-methylguanine-DNA methyltransferase (MGMT) inactivator. The drug had been tested in clinical trials to revert chemoresistance, but was associated with a low therapeutic index. A series of lomeguatrib conjugates with distinct alkyl chain lengths - i.e. C12, C14, C16, and C18 - was synthesized, and the MGMT depleting activity as well as cytotoxicity were determined on relevant mouse and human glioma cell lines. Drug-containing liposomes were prepared and characterized in terms of loading and in vitro release kinetics. The lipophilic lomeguatrib conjugates did not exert cytotoxic effects at 5 μM in the mouse glioma cell line and exhibited a similar MGMT depleting activity pattern as lomeguatrib. Overall, drug loading could be improved by up to 50-fold with the lipophilic conjugates, and the slowest leakage was achieved with the C18 derivative. The present data show the applicability of lipophilic lomeguatrib derivatization for incorporation into liposomes, and identify the C18 derivative as the lead compound for in vivo studies

Prominin-1 controls stem cell activation by orchestrating ciliary dynamics.

Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly-disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases